Ann Marie Fallon

The Biochemistry and Genetics of Mosquito Cells in Culture

Publisher Summary The importance of the mosquito as a vector of viral and protozoan pathogens and the increasing resistance of mosquitoes to insecticides provide a pressing rationale for intensified studies on the biochemistry and genetics of mosquito cells in culture. The relative expense of fetal calf serum has led various workers to test whether other types of serum would support growth of Aedes Albopictus cells. Characteristics allowing one to distinguish among lines derived from various mosquito (or other insect) species can be useful when several lines are maintained simultaneously in the same laboratory, and it is necessary to rule out cross-contamination. Ribosome biosynthesis requires specific assembly of macromolecules and precise coordination of the expression of genes which code for the ribosomal proteins.

Transient expression of the chloramphenicol acetyltransferase gene in cultured mosquito cells

A recombinant plasmid in which the bacterial chloramphenicol acetyltransferase (CAT) gene is under the control of the Drosophila heat-shock protein (hsp) 70 promoter has been introduced into cultured mosquito (Aedes albopictus) cells using 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) and dimethylsulfoxide (DMSO). CAT activity was induced by incubating transfected cells at 37 degrees C, and high levels of enzyme activity were maintained for more than 24 h after the temperature shock. Transfected DNA was maintained in the cells for at least 4 days. These experiments document an effective method for introducing purified DNA into cultured mosquito cells.

Factors affecting polybrene-mediated transfection of cultured Aedes albopictus (mosquito) cells☆

The polycation 1,5-dimethyl-1,5-diazaundecamethylene polymethobromide (polybrene) is superior to calcium phosphate for the introduction of purified DNA into cultured Aedes albopictus (mosquito) cells. Adsorption of the polybrene-DNA complex to mosquito cells was essentially linear for 6 h. However, the rate of adsorption of DNA increased when the DNA-polybrene mixture was preincubated for several hours prior to addition to cells. A recombinant plasmid carrying an inducible chloramphenicol acetyltransferase gene under the control of a Drosophila heat shock protein (hsp) promoter was used to show that expression of transfected DNA was highest when cells were treated with a freshly prepared polybrene-DNA mixture. Optimal expression was observed in cells transfected with 4-13 micrograms of DNA per 10(6) cells; transfection with 24 micrograms of DNA resulted in reduced CAT expression. Variation in the polybrene-DNA ratio improved transfection with high levels of DNA. In mosquito cells, CAT expression was independent of DNA methylation.

Methotrexate resistance in cultured mosquito cells

Abstract Several mosquito ( Aedes albopictus ) cell lines resistant to methotrexate, an inhibitor of the enzyme dihydrofolate reductase, have been characterized. On the basis of growth in the presence of methotrexate, the initial variants, Mtx-101, Mtx-102, Mtx-111, and Mtx-501, were 6.5-fold more resistant to methotrexate than the parental cells. From these cell lines, clones with increased resistance to methotrexate were selected. The second-level variants Mtx-5013 and Mtx-5011, were, respectively, 70- and 200-fold more resistant to methotrexate than wild type cells, and they contained 14- to 20-fold more dihydrofolate reductase activity. In extracts from Mtx-5013 and Mtx-5011 cells, the apparent affinity of dihydrofolate reductase for methotrexate was reduced about 20-fold with respect to that of wild type cells. Resistant cell lines became more sensitive to methotrexate during long-term passage in non-selective medium. This is the first description of methotrexate-resistant variants derived from cultured insect cells.

Identification of cDNAs corresponding to mosquito ribosomal protein genes

Sequences encoding mosquito (Aedes albopictus) ribosomal proteins L8, L14 and L31 were identified from a cDNA library made from size-selected polyadenylated mRNA. Candidate cDNAs corresponding to moderately abundant mRNAs were screened by translation of hybrid-selected transcripts in wheat-germ lysates. Translation products were extracted with acetic acid and analyzed by electrophoresis in two dimensions in the presence of unlabeled ribosomal proteins. The identity of translation products that coelectrophoresed with purified ribosomal protein standards was supported by peptide mapping. The cDNAs corresponding to L8 (pL8) and L31 (pL31) hybridized to cytoplasmic mRNAs of 1.4 and 0.9 kb, respectively. In Southern blots of genomic DNA digested with BamHI, HindIII or EcoRI, the cDNA inserts from both pL8 and pL31 gave simple hybridization patterns suggestive of a low copy number for mosquito ribosomal protein genes.

Isolation and characterization of puromycin-resistant clones from cultured mosquito cells

We have isolated from an established Aedes albopictus(mosquito) cell line clones which are resistant to the antibiotic puromycin. On the basis of growth and plating efficiency, clones Pur-8026 and Pur-8612 were five- and seven-fold more resistant, respectively, to puromycin than wild-type cells. In vitro protein synthesis was resistant to puromycin only in extracts prepared from Pur-8612 cells. Measurements of puromycin transport, crossresistance to colchicine, and sensitivity to Tween-80 indicated that resistance in Pur-8026 cells was due to membrane alteration(s) affecting permeability to puromycin. This is the first description of puromycin resistance in insect cells and also the first report of puromycin resistance in an animal cell variant associated with an alteration at the level of protein synthesis.

Isolation of ribosomal subunits from cultured mosquito cells

Abstract With cultured mosquito cells derived from Aedes albopictus , standard methods for preparation of ribosomal subunits have proved to be unsatisfactory. A new method has been developed for isolation of ribosomal subunits after treatment of mosquito cells with puromycin in vivo . Our results suggest that this method may be applicable to a variety of eukaryotic cell types.

Isolation and characterization of cycloheximide-resistant mosquito cell clones

Following treatment of cultured mosquito cells (Aedes albopictus line of Singh) with the chemical mutagen ethyl methanesulfonate, we were able to isolate three cycloheximide-resistant clones. On the basis of growth kinetics, plating efficiency, and protein synthesis, these clones are 10- to 30-fold more resistant to cycloheximide than the parent cells. Cell-free lysates made from these cells retained 30-65% of their endogenous protein synthesizing ability in the presence of cycloheximide concentrations as high as 300 micrograms/ml. Protein synthesis in lysates from the parental cells, however, is reduced to about 10% of the control value (i.e., in the absence of drug) at 14 micrograms of cycloheximide/ml and was completely abolished at 75 micrograms/ml. These results indicate that cycloheximide resistance in these cells is likely due to an alteration in the protein synthetic machinery. This is the first description of cycloheximide-resistant insect cells, and the best example of cycloheximide resistance in cells originating from a higher eukaryotic organism.

Evidence for multiple ribonucleases in crude extracts from cultured mosquito cells

Abstract Crude extracts from Aedes albopictus (mosquito) cells appear to contain several ribonuclease activities, which can be differentiated on the basis of heat-stability, pH optima and the effects of divalent cations. Ribonuclease activities which can be detected on polyacrylamide gels differ with respect to molecular weight, and various subcellular fractions appear to have distinct ribonuclease activities. Zinc chloride and heparin are effective inhibitors of ribonuclease activity in crude extracts. These results provide useful criteria for further characterization of the major ribonucleases present in cultured mosquito cells.

Phosphoribosylation of xanthine by extracts from insect cells

Abstract Using a sensitive TLC method, we have detected the production of xanthine monophosphate (XMP) from [ 14 C]xanthine by mosquito cell extracts incubated in the presence of phosphoribosyl pyrophosphate and a phosphatase inhibitor. Extracts from both cultured Aedes albopictus cells, and from intact Aedes aegypti mosquitoes contained activity; particularly high activity was found in extracts from adult male mosquitoes. XMP-producing activity was at least 4-fold higher in extracts from cultured mosquito cells than in extracts from Drosophila melanogaster Kc cells or Spodoptera frugiperda (Lepidoptera) cells.